Process for producing optically active amino compounds

ABSTRACT

The method for preparing an optically active (R)-amino compound characterized by the method comprising stereoselectively carrying out amino group transfer by action of an (R)-form-specific transaminase in the co-presence of a ketone compound (amino acceptor), and an amino compound (amino donor) of a racemic form or an (R)-form, to give an optically active (R)-amino compound. According to the present invention, it is made possible to easily prepare at a high yield the optically active (R)-amino compounds and the like having an aryl group and the like at their 1-position, which have been conventionally difficult to prepare.

This application is a continuation-in-part application of PCT/JP96/03054filed Oct. 21, 1996, the entire contents of which are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to optically active amino compounds havingan aryl group and the like at the 1-position, which can be used asintermediates for pharmaceuticals and agricultural chemicals.1-(3,4-Dimethoxyphenyl)-2-aminopropane, which is one of the desiredcompounds of the present invention, is an important compound asintermediates for CL316, 243 (J. D. Bloom et al., J. Med. Chem. 35,3081-3084 (1992)) and analogous compounds thereof, which are promisingas antidiabetics and agents for antiobesity under development.

In addition, (S)-2-amino-1-methoxypropane, which is another desiredcompound is a useful compound which can be used as intermediates forherbicides.

BACKGROUND ART

Processes for preparing optically active amino compounds having an arylgroup and the like at the 1-position by using an enzyme include a reportin Nakamichi et al. (Appl. Microbiol. Biotechnol., 33, 634-640 (1990))and Japanese Patent Examined Publication No. Hei 4-11194. It isdisclosed in these publications that an (S)-form can be efficientlyprepared by transferring an amino group to 1-(substitutedphenyl)-2-propanones by using an enzyme. Further, Japanese PatentExamined Publication No. Hei 4-11194 also discloses a preparation of an(R)-form; however, the present inventors have conducted additionalexperiment to examine microorganisms and substrate disclosed in JapanesePatent Examined Publication No. Hei 4-11194 and found that thereproducibility by means of this method is very poor, and thereby makingit difficult to use this method for practical purposes. Further,Stirling et al. disclose a method in which only the (S)-form isdecomposed by actions of an ω-amino acid transaminase to a racemic aminocompound produced by an organic reaction, to thereby obtain theremaining (R)-form (Japanese Patent Laid-Open No. Hei 3-103192).However, in this method, since the (S)-form is undesirably decomposed toobtain the (R)-form, the yield against the substrate is lowered to 50%or less. Accordingly, this method cannot be considered to beadvantageous from the aspect of costs. In addition, Stirling et al., theauthors as above, also disclose a method in which only (S)-aminocompounds are prepared from a ketone-form by using an ω-amino acidtransaminase in the presence of an amino donor. However, opticallyactive (R)-amino compounds cannot be produced by this method.

DISCLOSURE OF INVENTION

Accordingly, an object of the present invention is to provide a methodfor preparing optically active (R)-amino compounds by actions ofmicrobial enzymes efficiently and inexpensively.

The present inventors have found a microorganism from soil which canprepare optically active (R)-amino compounds with good yield by carryingout an amino group transfer to a ketone compound having an aryl group,and the like at 1-position (R)-form specifically and efficiently. Thismicroorganism has been deposited at the National Institute of Bioscienceand Human-Technology Agency of Industrial Science and Technology,located at 1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, Japan, andhas been given the depository designation number FERM BP-5228. Furtherstudies have been made on the reaction using this microorganism, and thepresent invention has been completed.

Specifically, the present invention, in essence, pertains to:

[1] A method for preparing an optically active (R)-amino compoundcharacterized by the method comprising stereoselectively carrying outamino group transfer by action of an (R)-form-specific transaminase inthe co-presence of a ketone compound (amino acceptor) represented by thefollowing general formula (I):

wherein n is 0 to 5; m is 0 or 1; and X represents an unsubstituted arylgroup having 6 to 14 carbon atoms, an aryl group having 6 to 14 carbonatoms and having one or more substituents selected from the groupconsisting of an alkyl group having 4 to 15 carbon atoms, a hydroxylgroup, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom,a nitro group, a carboxyl group, and a trifluoromethyl group, or amethoxyl group, and an amino compound (amino donor) of an achiral form,a racemic form, or an (R)-form represented by the general formula (II):

wherein X₁ and X₂ independently represent a hydrogen atom; astraight-chain alkyl group having 1 to 10 carbon atoms; a branched alkylgroup having 5 to 12 carbon atoms; an unsubstituted aryl group having 6to 14 carbon atoms; an aryl group having 6 to 14 carbon atoms and havingone or more substituents selected from the group consisting of an alkylgroup having 1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, achlorine atom, a bromine atom, an iodine atom, a nitro group, and acarboxyl group; an aralkyl group having 7 to 16 carbon atoms and havingone or more substituents selected from the group consisting of an alkylgroup having 1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, achlorine atom, a bromine atom, an iodine atom, a nitro group, and acarboxyl group; or a hydroxymethyl group or a hydroxyethyl group, togive an optically active (R)-amino compound represented by the generalformula (IV):

wherein n, m, and X have the same definitions as those of n, m, and X inthe general formula (I), respectively;

[2] The method for preparing an optically active (R)-amino compounddescribed in item [1] above, characterized in that in the generalformula (II), X₁ is an alkyl group having 2 to 10 carbon atoms, a phenylgroup, or a naphthyl group, and X₂ is an alkyl group having 1 or 2carbon atoms;

[3] The method for preparing an optically active (R)-amino compounddescribed in item [1] above, characterized in that the amino donorrepresented by the general formula (II) is an alkyl ester of D-alanine,the alkyl group having 1 to 8 carbon atoms;

[4] The method for preparing an optically active (R)-amino compounddescribed in item [1] above, wherein the amino donor represented by thegeneral formula (II) is (R)-1-phenylethylamine,(R)-1-naphthylethylamine, (R)-1-methylpropylamine, (R)-2-aminopentane,(R)-2-amino-1-propanol, (R)-1-methylbutylamine, (R)-1-phenylmethylamine,(R)-1-amino-1-phenylethanol, (R)-2-amino-2-phenylethanol,(R)-3-aminoheptane, (R)-1-amino-3-phenylpropane,(R)-2-amino-4-phenylbutane, (R)-2-amino-3-phenylpropanol,(R)-3,4-dimethoxyaminopropane, (R)-1-methylheptylamine, benzylamine,(S)-2-phenylglycinol, 3-aminophenylbutane, L-phenylalaninol,(R)-2-amino-1-methoxypropane, D-alanine methyl ester, D-alanine ethylester, or a racemic compound thereof;

[5] The method for preparing an optically active (R)-amino compounddescribed in any one of items [1] to [4] above, characterized in that inthe general formula (I) and the general formula (IV), n and m are n=1and m=0;

[6] The method for preparing an optically active (R)-amino compounddescribed in any one of items [1] to [5] above, characterized in that inthe general formula (I) and the general formula (IV), X is phenyl,2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2,4-dimethoxyphenyl,3,4-dimethoxyphenyl, 3-trifluoromethylphenyl, or methoxyl;

[7] The method for preparing an optically active (R)-amino compounddescribed in any one of items [1] to

[6] above, characterized in that the amino acceptor represented by thegeneral formula (I) and the amino donor represented by the generalformula (II) are brought into contact with a culture of microorganisms,separated bacterial cells, treated bacterial cells, or immobilizedbacterial cells which produce (R)-form-specific transaminases;

[8] The method for preparing an optically active (R)-amino compounddescribed in any one of items [1] to [6] above, characterized in thatthe amino acceptor represented by the general formula (I) and the aminodonor represented by the general formula (II) are brought into contactwith cell-free extracts of microorganisms, crudely purified enzymes,purified enzymes, or immobilized enzymes which produce (R)-form-specifictransaminases;

[9] The method for preparing an optically active (R)-amino compounddescribed in any one of items [1] to [8] above, wherein themicroorganism for producing the transaminase is a microorganismbelonging to the genus Arthrobacter;

[10] The method for preparing an optically active (R)-amino compounddescribed in item [9] above, wherein the microorganism belonging to thegenus Arthrobacter is Arthrobacter species (Arthrobacter sp.) KNK168(FERM BP-5228);

[11] The method for preparing an optically active (R)-amino compounddescribed in any one of items [1] to [10] above, characterized by addingto a medium, when culturing the microorganism for producing thetransaminase, one or more members selected from the group consisting of(RS)-1-methylpropylamine, (RS)-1-phenylethylamine,(RS)-1-methylbutylamine, (RS)-3-amino-2,2-dimethylbutane,(RS)-2-amino-1-butanol, and (R)- or(RS)-1-(3,4-dimethoxyphenyl)aminopropane as an inducer for the enzyme;

[12] The method for preparing an optically active (R)-amino compounddescribed in item [1] above, characterized by carrying out the reactionat a pH of not less than 5 and not more than 12 in the amino grouptransfer reaction;

[13] The method for preparing an optically active (R)-amino compounddescribed in item [1] above, characterized by adding a surfactant or afatty acid as a reaction accelerator upon reaction in the amino grouptransfer reaction;

[14] A method for preparing an optically active (S)-amino compound,characterized by the method comprising stereoselectively carrying outamino group transfer reaction by action of an (R)-form-specifictransaminase to an amino compound of a racemic form represented by thegeneral formula (V) in the presence of a ketone compound (aminoacceptor) represented by the general formula (III), to give an opticallyactive (S)-amino compound represented by the general formula (VI).

[15] An (R)-form-specific transaminase obtainable from a culture of amicroorganism belonging to the genus Arthrobacter; and

[16] The (R)-form-specific transaminase described in item [15] above,wherein the transaminase contains an amino acid sequence of:

Glu-Ile-Val-Tyr-Thr-His-Asp-Thr-Gly-Leu-Asp-Tyr (SEQ ID NO:1) in theneighborhood of an amino terminal of the enzyme protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the optimal pH for the enzyme of the presentinvention.

FIG. 2 is a graph showing the stable pH for the enzyme of the presentinvention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in detail below. First, thereaction scheme in the present invention is shown as follows.

The present inventors have carried out repeated screening to separatebacteria from domestic soil which have an ability of producing (R)-aminocompounds in (R)-form selectivity using ketones as substrates, andconsequently have found that bacteria belonging to the genusArthrobacter have strong activity for catalyzing this reaction. Amongthem, the bacteriological natures of Arthrobacter sp. KNK168 (FERMBP-5228), a typical example thereof, are shown as follows.

Cell Morphology Rod (Coryne type) Gram Staining Positive Spore FormationNone Motility None Colony Morphology Round, regular, entire, yellow,smooth, glossy, semi-translucent, convex, 2 m in diameter (Bennett'sagar medium) Growth (30° C.) + (37° C.) − Catalase + Oxidase − OF Test(glucose) − (oxidative) Cell Wall No mycolic acid; diamino acid islysine. Fatty Acid Analysis Almost all the acids present arethree-branched iso and anteiso acid.

From the aspects that an (R)-form amine, such as (R)-1-phenylethylamine,is used as an amino donor and that the resulting product is an opticallyactive (R)-amino compound as described in detail below, the transaminaseproduced by this bacterium is obviously different from an enzyme ofBrevibacterium linens IFO12141 used in Nakamichi et al. (Appl.Microbiol. Biotechnol., 33, 634-640 (1990)) and an ω-amino acidtransaminase used in Stirling et al. (Japanese Patent Laid-Open No. Hei3-103192), in which an (S)-amine is used as an amino donor and theresulting product is an optically active (S)-amino compound.

Further, the transaminase derived from Arthrobacter which can be used inthe present invention is different from the above-mentioned enzymederived from Brevibacterium in many other aspects. For example, thereare differences from the aspect that inorganic ammonium salts such asammonium chloride, and L-amino acids (being (S)-amines) such as glutamicacid and aspartic acid cannot be used as an amino donor (Table 1).

TABLE 1 Transami- Enzyme nase of Used in Amino Group Present NakamichiDonor Invention et al. Inorganic − + Ammonium Salt L-Amino Acid − +

In addition, when the transaminase derived from Arthrobacter which canbe used in the present invention is compared with the ω-amino acidtransaminase used in Stirling et al. (Japanese Patent Laid-Open No. Hei3-103192), in addition to the obvious difference regarding theabove-mentioned substrate specificity, there are differences from theaspect that the transaminase derived from Arthrobacter does not act toω-amino acids such as β-alanine and 4-aminobutyric acid, and ω-aminessuch as n-butylamine and putrescine, and DL-3-aminobutyric acid, and thelike, and that the transaminase derived from Arthrobacter is affectedonly a little by reaction inhibitors such as hydroxylamine andphenylhydrazine.

In addition, as other enzymes similar to the enzyme used by the presentinventors, benzylamine transaminase is disclosed in Okada et al.(Japanese Patent Laid-Open No. Hei 6-178685), wherein the benzylaminetransaminase strongly acts to benzylamine in the presence of pyruvicacid to form L-alanine and benzaldehyde. However, the benzylaminetransaminase has a similar optical specificity to that of theabove-mentioned enzyme derived from Brevibacterium or to that of theω-amino acid transaminase from the aspect that the optical activity ofthe product by the transaminase action of this enzyme is that ofL-alanine of an (S)-form, and it can be said that the benzylaminetransaminase is completely different from the enzyme derived fromArthrobacter which can be used in the present invention.

Further, when these enzymes are compared, it seems that there are somedifferences in influence caused by reaction inhibitors such asphenylhydrazine, D-penicillamine, and p-chloromercuribenzoic acid.

The differences with the ω-amino acid transaminase derived fromPseudomonas sp. F-1 (Agric. Biol. Chem., 42, 2363-2367 (1978), Agric.Biol. Chem., 43, 1043-1048 (1979), J. Biol. Chem., 258, 2260-2265(1983)) which have been well studied among the ω-amino acidtransaminases, and with the benzylamine transaminase of Okada et al.(Japanese Patent Laid-Open No. Hei 6-178685) are summarized and shown inTable 2. In a case of the transaminase action in the present invention,the data of the purified enzyme are used, and in cases of those by othertwo enzymes, the data of the purified enzymes which are disclosed ineach of the literature publication and patent publication are used.Pyruvic acid is used as an amino acceptor in all cases.

TABLE 2 Transaminase Transaminase of Present ω-Amino Acid of OkadaInvention Transaminase et al. Substrate (Relative (Relative (RelativeSpecificity Activity: %) Activity: %) Activity: %) β-Alanine 0 100 04-Aminobutyric 0 40 acid DL-3- 0 96 Aminobutyric acid n-Butylamine 0 600 Benzylamine 0.8 45 100 Putrescine 0 55 0 β-Phenethylamine 0 54 9 AminoGroup Donor L-Alanine — + + Inhibitor (Relative (Relative (Relative (1mM each) Activity: %) Activity: %) Activity: %) (without 100 100 100addition) Hydroxylamine 17 0 30 (0.1 mM) Phenylhydrazine 82 6 27D-Penicillamine 93 65 0 p-Chloromercuri- 53 100 9 benzoic acid (0.1 mM)CuSO₄ 44 5 (0.5 mM) Gabaculine 24 0

The activity of the transaminase (intracellular enzyme) which can beused in the present invention against the typical substrates of the(ω-aminotransferases is shown in Table 3. In the case of using anω-amino acid or an ω-amine as an amino donor, the ω-aminotransferaseactivity is extremely low, and the transferase used in the presentinvention does not act to β-alanine which is a typical substrate ofω-amino acid-pyruvic acid aminotransferase. The transferase used in thepresent invention shows an especially high activity to(R)-1-phenylethylamine. Therefore, it is quite different from theω-amino transaminase in the substrate specificity.

TABLE 3 Relative Amino Donor Amino Acceptor Activity (%) β-AlaninePyruvic acid 0 4-Aminobutyric 2-Ketoglutaric 0 acid acid2,5-Diaminovalerate 2-Ketoglutaric 2 (α,ω-amino acid) acid DL-Ornithine2-Ketoglutaric 0 acid DL-Lysine 2-Ketoglutaric 0 acid Putrescine2-Ketoglutaric 0 (α,ω-Diamine) acid α-2,4-Diamino- Pyruvic acid 0butyric acid Taurine 2-Ketoglutaric 0 acid DL-Asparagine 2-Ketoglutaric0 acid DL-Glutamine 2-Ketoglutaric 0 acid (R)-1-Phenylethyl- Pyruvicacid 100 amine (Control)

The (R)-form-specific transaminase produced by Arthrobacter species(Arthrobacter sp.) KNK168 (FERM BP-5228) used in the present inventionhas an amino acid sequence of SEQ ID NO:1 in Sequence Listing:

Glu-Ile-Val-Tyr-Thr-His-Asp-Thr-Gly-Leu-Asp-Tyr in the neighborhood ofan amino terminal of the enzyme protein.

In a case where the amino group transfer action which can be used in thepresent invention is carried out, the enzymatic activity is increased byaddition of an inducer upon culturing microorganisms. The above inducersinclude one or more members of amines, such as (RS)-1-methylpropylamine,(RS)-1-phenylethylamine, (RS)-1-methylbutylamine,(RS)-3-amino-2,2-dimethylbutane, (RS)-2-amino-1-butanol, and (R)- or(RS)-1-(3,4-dimethoxyphenyl)aminopropane.

The transaminase derived from Arthrobacter which can be used in thepresent invention can be used in various forms. In other words, not onlycultured microorganisms, separated bacterial cells and treated bacterialcells, but also cell-free extracts, crudely purified enzymes, purifiedenzymes, and the like can be used. Further, immobilized products ofthese cells, immobilized products of the treated bacterial cells,immobilized products of enzyme proteins to immobilizing carriers (forexample, anionic exchange resins) and the like can be also used. Here,the immobilization can be carried out by conventional methods (forexample, Japanese Patent Laid-Open No. Sho 63-185382).

Supporting materials which can be used in the immobilization includevarious kinds of anionic exchange resins, such as various amines,ammonium salts and diethanolamine type resins having functional groups.Suitable examples thereof include phenol-formaldehyde anionic exchangeresins such as Duolite A568 and DS17186 (registered trademark of Rohmand Haas Company); polystyrene resins, such as Amberlite IRA935, IRA945,and IRA901 (registered trademark of Rohm and Haas Company), LewatitOC1037 (registered trademark of Bayer A. G.), and Diaion EX-05(registered trademark of MITSUBISHI KASEI CORPORATION), and the like. Inaddition, supporting materials, such as DEAE-cellulose, can be used.

Further, cross-linking agents are usually used in order to make theadsorption of enzyme more firm and stable. A preferable example of thecross-linking agents includes glutaraldehyde. The enzymes used includepurified enzymes, as well as enzymes at various levels of thepurification, such as partially purified enzymes, solutions of disruptedcells, and cell-free extracts.

As a preparation method of the immobilized enzyme, conventionalpreparation methods may be employed, including, for example, a methodwherein the cross-linking treatment is carried out after adsorption ofan enzyme to a supporting material.

In other words, in the present invention, it is preferable that theamino acceptor represented by the general formula (I) and the aminodonor represented by the general formula (II) are brought into contactto a culture of microorganisms, separated bacterial cells, treatedbacterial cells, or immobilized bacterial cells which produce(R)-form-specific transaminase, or into contact to cell-free extracts ofmicroorganisms, crudely purified enzymes, purified enzymes, orimmobilized enzymes which produce (R)-form-specific transaminases.

The amino acceptors which can be used in the present invention includeketones represented by the general formula (I). In the formula, n is 0to 5; m is 0 or 1; and X represents an unsubstituted aryl group having 6to 14 carbon atoms, an aryl group having 6 to 14 carbon atoms and havingone or more substituents selected from the group consisting of an alkylgroup having 4 to 15 carbon atoms, a hydroxyl group, a fluorine atom, achlorine atom, a bromine atom, an iodine atom, a nitro group, a carboxylgroup, and a trifluoromethyl group, or a methoxy group.

Among them, a compound with n=1 and m=0, for example, 1-aryl-2-propanoneor 1-methoxy-2-propanone, is preferable. In addition, the compound inwhich the aryl group is a phenyl group, a 2-methoxyphenyl group, a3-methoxyphenyl group, a 4-methoxyphenyl group, a 2,4-dimethoxyphenylgroup, a 3,4-dimethoxyphenyl group, or a 3-trifluoromethylphenyl groupis also preferable.

The amino donors used in the present invention include amino compoundsrepresented by the general formula (II) of an achiral form, a racemicform, or an (R)-form. In the general formula (II), X₁ and X₂independently represent a hydrogen atom; a straight-chain alkyl grouphaving 1 to 10 carbon atoms; a branched alkyl group having 5 to 12carbon atoms; an unsubstituted aryl group having 6 to 14 carbon atoms;an aryl group having 6 to 14 carbon atoms having one or moresubstituents selected from the group consisting of an alkyl group having1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, a chlorine atom,a bromine atom, an iodine atom, a nitro group, and a carboxyl group; anaralkyl group having 7 to 16 carbon atoms and having one or moresubstituents selected from the group consisting of an alkyl group having1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, a chlorine atom,a bromine atom, an iodine atom, a nitro group, and a carboxyl group; ora hydroxymethyl group or a hydroxyethyl group.

Among the above-mentioned amines, amines in which X₁ is an alkyl grouphaving 2 to 10 carbon atoms, a phenyl group or a naphthyl group, and X₂is an alkyl group having 1 or 2 carbon atoms, or alkyl esters ofD-alanine, the alkyl group having 1 to 8 carbon atoms, are particularlypreferable. Concrete examples of the amines include, for instance,(R)-1-phenylethylamine, (R)-1-naphthylethylamine,(R)-1-methylpropylamine, (R)-2-aminopentane, (R)-2-amino-1-propanol,(R)-1-methylbutylamine, (R)-1-phenylmethylamine,(R)-1-amino-1-phenylethanol, (R)-2-amino-2-phenylethanol,(R)-3-aminoheptane, (R)-1-amino-3-phenylpropane,(R)-2-amino-4-phenylbutane, (R)-2-amino-3-phenylpropanol,(R)-3,4-dimethoxyaminopropane, (R)-1-methylheptylamine, benzylamine,(S)-2-phenylglycinol, 3-aminophenylbutane, L-phenylalaninol,(R)-2-amino-1-methoxypropane, D-alanine methyl ester, D-alanine ethylester, and the like.

The concentrations of the substrates used in the reaction are asfollows. It is preferred that the concentration of the amino acceptor isfrom 0.1 to 10%, preferably from 3 to 5%. Regarding the concentration ofthe amino donor, it is preferred that the concentration of an (R)-form,in the case of chiral amine, is about 80 to 150 mol % to the aminoacceptor. In addition, racemic amino compounds can be also used as aminodonors. In such cases, however, twice the concentration as that of the(R)-form is needed.

The pH during the reaction is usually a pH of from 5.0 to 12.0,preferably a pH of from 7.0 to 10.0. The temperature during the reactionis usually from 25° to 40° C., preferably from 30° to 35° C.

Further, the yield of reaction may be increased by adding a surfactant,including sodium dodecyl sulfate (SDS), Triton X-100, cetyltrimethylammonium bromide (CTAB), or the like, or a fatty acid,including linoleic acid, oleic acid, or the like, in an amount of from0.1 to 10%.

The optically active amino compounds which are prepared by the method ofthe present invention are the (R)-amino compounds represented by thegeneral formula (IV). In the formula, n, m, and X, respectively, has thesame definitions as those in the general formula (I).

Concrete examples of the compounds include, for instance,

(R)-1-phenyl-2-aminopropane,

(R)-1-phenyl-3-aminobutane,

(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane,

(R)-3-(trifluoromethylphenyl)-2-aminopropane,

(R)-3-(p-methoxyphenyl)-2-aminopropane,

(R)-4-(p-methoxyphenyl)-2-aminobutane,

(R)-4-(3′,4′-methylenedioxyphenyl)-2-aminobutane,(R)-4-(p-hydroxyphenyl)-2-aminobutane,

(R)-1-(3-trifluoromethylphenyl)-2-aminopropane,

(R)-2-amino-1-methoxypropane, and the like.

By the preparation method of the present invention, for example, in acase where 1-(3,4-dimethoxyphenyl)-2-propanone used as an amino acceptorand (R)-1-phenylethylamine used as an amino donor, each having aconcentration of 3%, are reacted by using Arthrobacter species KNK168(FERM BP-5228) for about 20 hours, 75% or more of1-(3,4-dimethoxyphenyl)-2-propanone can be converted to(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane with an optical purity of 99%ee or more by the amino group transfer action.

The quantitative analysis of the optically active amino compoundobtained as the reaction product can be carried out by means ofhigh-performance liquid chromatography. For example, the quantitativeanalysis can be carried out by separating the reaction mixture by usinga reversed phase column (Cosmosil 5C₁₈-AR, NACALAI TESQUE, INC., and thelike) and using 25% acetonitrile as a moving phase, and the detectedabsorptions at 210 nm were compared with the control. In addition, thereare several methods for measuring the optical purity. For example, theanalysis of the optical purity can be carried out by binding theresulting optically active amino compound to N-carboxy-L-leucineanhydride, and the like, in order to form a diastereomer, and thereafterseparating and quantitatively analyzing this diastereomer by theabove-mentioned high-performance liquid chromatography method.

The method for separating the optically active amino compound, thedesired compound, after the reaction can be carried out by theconventional method in which extraction by an organic solvent anddistillation are combinably used. For example, ethyl acetate, toluene,and the like can be used as the extraction solvent. First, the reactionsolution is made acidic and the ketones are removed therefrom byextraction. Thereafter, the reaction mixture is made alkaline, andseparate the amines including the desired compound by extraction.Further, by further means of distillation of the extracted fraction, thedesired optically active amino compound can be isolated.

In the transaminase which can be used in the present invention, whenacted the racemic amino compound represented by the general formula (V)in the presence of the ketone compound (amino acceptor) represented bythe general formula (III), only its (R)-form is selectively converted toketone compounds, because the transaminase only acts to the (R)-form, sothat the (S)-form remains unchanged, and thus, collected as the aminocompound. Therefore, the (S)-amino compounds represented by the generalformula (VI) can be easily prepared from racemic amino compounds byusing the transaminase of the present invention.

For example, in a case where racemic forms of1-(3,4-dimethoxyphenyl)-2-aminopropane or 2-amino-1-methoxypropane arereacted with pyruvic acid used as an amino acceptor using Arthrobacterspecies KNK168 (FERM BP-5228), the (R)-form amino compound is convertedto the ketone form by the amino group transfer, so that the (S)-formamino compound can be obtained at about 50% yield in a concentratedstate up to a high optical purity level.

The present invention will be described in further detail by means ofthe working examples, but the scope of the present invention is by nomeans limited to these examples.

EXAMPLE 1

Each 2 g of soil samples collected at various places in this country wassuspended in 5 ml of physiological saline. 0.2 ml of the supernatantthereof was added to 4 ml of an S medium (2 g/L KH₂PO₄, 2 g/L K₂HPO₄,0.3 g/L MgSO₄.7H₂O, 5 g/L glycerol, 3 g/L NaCl, 1 g/L yeast extractpowder, 0.004 g/L FeSO₄.7H₂O, 0.0005 g/L ZnSO₄.7H₂O, 0.0005 g/LMnCl₂.4H₂O (pH 7.5); and 2-oxoglutaric acid or pyruvic acid, filtratedby a microorganisms exclusion filter after a treatment of autoclaving,and (R)-1-(3,4-dimethoxyphenyl)-2-aminopropane being added so as to givefinal concentrations of 1.5 g/L and 1.0 g/L, respectively). Theresulting culture was subjected to an enrichment culture at 30° C. for 3to 7 days. Each 0.2 ml of the culture in which the bacteria were grownwas spread on an S-medium plate containing 1.5% of agar, and thecolonies were grown by culturing at 30° C. The grown colonies weresubjected to shaking culture in the S-medium. After harvesting thecells, they were suspended in 0.25 ml of a reaction mixture containing a0.1 M carbonic acid buffer (pH 8.5), 50 mM pyruvic acid and 30 mM(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane, and the components werereacted with stirring at 30° C. for 24 hours.

The resulting reaction mixture was separated by thin-layerchromatography (Kieselgel 60F254 (Merck); developing solvent beingdiethyl ether:methanol:aqueous ammonia solution (27%)=50:50:2). Thedecrease of the substrates was detected by ninhydrin, and the formationof the resulting product, 1-(3,4-dimethoxyphenyl)-2-propanone, wasdetected by 0.4% of 2,4-dihydrophenyl hydrazine. With regard to thestrains in which the formation of the products was confirmed, thepresence or absence of the (R)-form-specific amino group transfer actionactivity was examined by carrying out its reverse reaction.Specifically, the cultured cells were added to a reaction mixturecontaining 0.6% of 1-(3,4-dimethoxyphenyl)-2-propanone and 0.6% of(R)-1-phenylethylamine, and the components were reacted at 30° C. for 2days. As a result, the Arthrobacter species KNK168 strain was found tohave the (R)-form-specific amino group transfer activity.

EXAMPLE 2

When culturing the Arthrobacter species KNK168 strain by the methoddescribed in Example 1, (RS)-1-methylpropylamine,(RS)-1-methylbutylamine, (RS)-3-amino-2,2-dimethylbutane,(RS)-2-amino-1-butanol, or (RS)-1-phenylethylamine was added to themedium in place of (R)-1-(3,4-dimethoxyphenyl)-2-aminopropane ((R)-DMA).The cells were separated from the medium, and the reaction was carriedout at 30° C. for 20 hours using the above cells in a reaction mixturecontaining 1% of 1-(3,4-dimethoxyphenyl)-2-propanone and 1% of(RS)-1-methylpropylamine. As a result, an increase of the reactivity wasobserved as shown in Table 4.

TABLE 4 (R)-DMA Formed Inducer (%) (RS)-1-Methylpropylamine 22.3(RS)-1-Methylbutylamine 13.0 (RS)-1-Phenylethylamine 8.0(RS)-3-Amino-2,2-dimethylbutane 18.6 (RS)-2-Amino-1-butanol 12.9 (R)-DMA0.9 No Addition 0.3

EXAMPLE 3

The Arthrobacter species KNK168 strain was inoculated to an R medium (5g/L KH₂PO₄, 5 g/L K₂HPO₄, 3 g/L NaCl, 1 g/L MgSO₄.7H₂O, 0.005 g/LFeSO₄.7H₂O, 0.001 g/L ZnSO₄.7H₂O, 0.001 g/L MnCl₂.4H₂O (pH 7.5), 15 g/Lglycerol, 2 g/L yeast extract powder, 8 g/L PRO-EX (BANSYU CHOMIRYO CO.,LTD.); pyruvic acid, filtrated by a microorganisms exclusion filterafter a treatment of autoclaving, and (RS)-1-methylpropylamine beingadded so as to give final concentrations of 1.6 g/L and 2.0 g/L,respectively, and being adjusted to pH 7.2), and then subjected toshaking culture at 30° C. for 24 hours. The cultured cells wereharvested by centrifugation, and the harvested cells were suspended in areaction mixture (pH 8.5) containing 1% of1-(3,4-dimethoxyphenyl)-2-propanone, 0.1% of Triton X-100, and 1% of anamino donor shown in Table 2. The components were reacted at 35° C. for20 hours. As shown in Table 5, it was found that(RS)-1-phenylethylamine, (RS)-1-methylbutylamine,(RS)-1-methylpropylamine, (RS)-2-aminopentane, and the like, functionedas amino donors, among which (RS)-1-phenylethylamine was most highlypreferable.

TABLE 5 (R)-DMA Formed Amino Group Donor (%) (RS)-1-Phenylethylamine61.3 (RS)-1-Methylbutylamine 54.7 (RS)-1-Methylpropylamine 28.2(RS)-2-Aminopentane 56.9 (RS)-2-Amino-1-propanol 9.6(RS)-2-Amino-2-phenylethanol 48.4 (RS)-2-Amino-3-phenylpropanol 22.7(RS)-2-Amino-4-phenylbutane 27.6 (RS)-1-Amino-2-phenylpropane 1.8(RS)-1-Phenylmethylamine 3.1 (RS)-3-Aminoheptane 7.4(RS)-1-Naphthylethylamine 34.2 D-Alanine methyl ester 3.5 D-Alanineethyl ester 4.2 D-Alanine 3.0

EXAMPLE 4

The cells obtained by culturing the Arthrobacter species KNK168 strainin the R medium were suspended in a reaction mixture containing 2% of1-(3,4-dimethoxyphenyl)-2-propanone and 4% of (RS)-1-phenylethylamine.The surfactants and fatty acids shown in Table 6 were added thereto, andthe mixture was reacted at 30° C. at a pH of 8.5 for 20 hours. As aresult, it was found that the rate of reaction was increased by additionof sodium dodecyl sulfate, linoleic acid, oleic acid, or the like to thereaction mixture.

TABLE 6 (R)-DMA Formed Additives (%) (%) Triton X-100 0.1 31.6 0.5 34.0Sodium dodecyl sulfate 0.1 45.3 0.5 42.2 1.0 46.3 2.0 50.1 3.0 51.9 4.053.1 Cetyl trimethyl ammonium 0.1 29.1 bromide (CTAB) Linoleic Acid 2.945.1 Oleic Acid 2.9 44.9 (No Addition) 28.1

EXAMPLE 5

Using the cells obtained by culturing the Arthrobacter species KNK168strain in the R medium, the components were reacted at 35° C. for 20hours, while making the pH of a reaction mixture variable from 7.5 to9.5, the reaction mixture containing 2% of1-(3,4-dimethoxyphenyl)-2-propanone, 1.4% of (RS)-1-phenylethylamine,and 0.1% of sodium dodecyl sulfate. As a result, the rate of reaction ata pH of 8.5 to 9.0 was favorable as shown in Table 7.

TABLE 7 (R)-DMA Formed Reaction pH (%) 7.5 16.8 8.0 30.8 8.5 40.0 9.041.6 9.5 36.1

EXAMPLE 6

30 ml of overnight preculture of the Arthrobacter species KNK168 strainwas inoculated to 1.5 liter of a J medium (5 g/L KH₂PO₄, 5 g/L K₂HPO₄, 1g/L NaCl, 1 g/L MgSO₄.7H₂O, 0.005 g/L FeSO₄.7H₂O, 0.001 g/L ZnSO₄.7H₂O,0.001 g/L MnCl₂.4H₂O, 0.0005 g/L CuSO₄.5H₂O (pH 7.5), 40 g/L glycerol, 3g/L yeast extract powder, 20 g/L PRO-EX (BANSYU CHOMIRYO CO., LTD.), apH being adjusted to 7.5) in a 2 liter-mini jar, and the bacterium wascultured at 30° C. at 0.5 vvm at 450 rpm for 43 hours while keeping andadjusting the culture to a pH of 7.5. Incidentally, from the start ofculturing, (RS)-1-methylpropylamine filtered by a microorganismsexclusion filter was added so as to have a final concentration of 4 g/Lafter 14 hours. The final cell concentration (OD₆₁₀) was about 29 at theend of the culture, and the transaminase activity was 0.3 unit per theculture at that time. Incidentally, the unit of enzymatic activity meansthe intensity of the enzymatic activity which converts a 1 μM substrateof 1-(3,4-dimethoxyphenyl)-2-propanone to(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane at 30° C. in 1 minute.

The cells harvested from 1.5 liter of the culture were suspended in areaction mixture (pH 8.5) containing 45 g of1-(3,4-dimethoxyphenyl)-2-propanone, 28.3 g of (R)-1-phenylethylamine,and 65.0 g of oleic acid. The components were reacted at 30° C. for 39hours. As a result, 81.6% of the substrate was converted to(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane. After the pH of the reactionmixture to 2.0 with hydrochloric acid was adjusted, the reaction mixturewas extracted with toluene to separate ketones by migrating the ketonesto the organic layer. After the pH of the aqueous layer was adjusted to12 with sodium hydroxide, the reaction mixture was extracted withtoluene again, and 32.9 g of (R)-1-(3,4-dimethoxyphenyl)-2-aminopropanewas contained in the organic layer. The separation of the desiredcompound from the extract was carried out by distillation. As a result,30.3 g of (R)-1-(3,4-dimethoxyphenyl)-2-aminopropane was contained inthe main distillated fraction. The total yield obtained from thesequential procedures was about 68.7%, and the optical purity was about99.6% ee ((R)-form).

EXAMPLE 7

The Arthrobacter species KNK168 strain was subjected to shaking culturein a Sakaguchi flask containing 400 ml of the R medium at 30° C. for 30hours. After harvesting the cells, the cells were suspended in 40 ml ofa 20 mM potassium phosphate buffer (pH 6.8) containing 0.1% of2-mercaptoethanol. After the cells were disrupted by ultrasonic sound,the precipitates were removed by centrifugation to obtain 34 ml of acell-free extract obtained as a supernatant.

Using 10 ml of the cell-free extract, the components were reacted in 100ml of a reaction mixture comprising a 0.1 M Tris-hydrochloric acidbuffer (pH 8.5) containing 0.5 g of 1-(3,4-dimethoxyphenyl)-2-propanoneand 0.34 g of (R)-1-phenylethylamine at 30° C. for 24 hours. As aresult, 0.36 g of (R)-(3,4-dimethoxyphenyl)-2-aminopropane was formed.

EXAMPLE 8

The Arthrobacter species KNK168 strain was cultured in the same manneras in Example 7, and the cells were separated from 1 ml of the culture.The cells were suspended in 1 ml of a reaction mixture containing a 0.1M Tris-hydrochloric acid buffer (pH 8.5), 40 mg of racemic1-(3,4-dimethoxyphenyl)-2-aminopropane, 20 mg of pyruvic acid, and 2% ofsodium dodecyl sulfate. The components were reacted at 30° C. for 48hours with stirring. As a result of the analysis of the mixture afterthe reaction, 20.5 mg of 1-(3,4-dimethoxyphenyl)-2-aminopropaneremained, and its optical purity was 96% ee ((S)-form).

EXAMPLE 9

The Arthrobacter species KNK168 strain was cultured in 3.5 liter of theJ medium at 30° C. for 43 hours using a 5 liter mini-jar in the samemanner as in Example 6. The cells were harvested by centrifugation, andthe harvested cells were suspended in 1 liter of 20 mM potassiumphosphate buffer (pH 6.8) containing 0.01% of 2-mercaptoethanol. Thecells were disrupted by Dynomill (Trade Mark, Switzerland), and 810 mlof the supernatant was separated by centrifugation. Protamine sulfatewas added to this supernatant so as to give a concentration of 50 mg/ml,and nucleic acids were removed. Ammonium sulfate was added thereto so asto have a concentration of 30% to saturation, and the precipitatedprotein was removed. Thereafter, ammonium sulfate was added again so asto have a concentration of 60% to saturation, and the precipitatedprotein was separated. This protein was dissolved in the above-mentionedbuffer, and it was dialyzed against the same buffer. After thecomposition of the buffer was adjusted to 20% (v/v) of glycerol, 0.3 Mof NaCl, and 20 μM of pyridoxal phosphate, a DEAE-Sepharose, Fast-Flow(Pharmacia) column (φ4.4 cm×20 cm) was charged with the buffer, whichwas eluted with an NaCl linear concentration gradient of from 0.3 to 0.5M. After this active fraction was collected and dialyzed, ammoniumsulfate was added thereto so as to give a concentration of 0.2 M. APhenylsepharose (Pharmacia) column (φ2.2 cm×17 cm) was charged with theactive fraction, which was eluted with an ammonium sulfate linearconcentration gradient of from 0.2 to 0 M. After the concentration ofthe ammonium sulfate in the active fraction was adjusted to 0.6 M,Butylsepharose (Pharmacia) column (φ2.2 cm×17 cm) was charged with theactive fraction, which was eluted with an ammonium sulfate linearconcentration gradient of from 0.6 to 0.2 M. The active fraction wascollected and concentrated by ultrafiltration. Thereafter, theconcentrated fraction was subjected to electrophoresis withSDS-polyacrylamide gel, and as a result, substantially a single band wasformed at the position corresponding to a molecular weight of about37,000.

EXAMPLE 10

The amino acid sequence in the neighborhood of the amino terminal of thepurified enzyme protein obtained in Example 9 was analyzed by aGas-Phase Protein Sequencer (470A, Applied Biosystems, Inc.). As aresult, it was found that the above enzyme had an amino acid sequence ofSEQ ID NO:1 in Sequence Listing:

Glu-Ile-Val-Tyr-Thr-His-Asp-Thr-Gly-Leu-Asp-Tyr in the neighborhood ofthe amino terminal end.

EXAMPLE 11

Using the purified enzyme obtained in Example 9, reactivities to variousamino compounds when using pyruvic acid as an amino acceptor wereevaluated. 150 μl of a solution of the purified enzyme diluted ten-foldswas added to 150 μl of a substrate solution (0.1 M potassium phosphatebuffer (pH 8.3), 40 mM pyruvic acid, 0.1 mM pyridoxal phosphate, and 40mM various amino compounds). The components were reacted at 30° C. for 1hour. The reaction tube was transferred into boiling water to stop thereaction, and, thereafter, a part of the reaction mixture was dilutedfive-folds. Ten μl of 0.1 M boric acid buffer (pH 8.0) and 20 μl of anethanol solution of 80 mM of 4-fluoro-7-nitro-2,1,3-benzoxadiazole(NBD-F) were added to 10 μl of the above-mentioned diluted reactionmixture. The components were reacted at 60° C. for 1 minute, and,thereafter, the reaction mixture was ice-cooled, and 460 μl of 5 mM HClwas added thereto. This reaction mixture was analyzed byhigh-performance liquid chromatography using fine pack C18-5 column andusing a 0.1 M potassium phosphate buffer (pH 6.5) and CH₃CN (90:10) asan eluent solution to detect formation of alanine-NBD using excitationwavelength of 470 nm and using detection wave of 530 mm. The resultsthereof are shown in Table 8 in terms of relative activity in the caseof using R-1-phenylethylalanine as a substrate.

It is apparent from the Table 8 that (S)-2-phenylglycinol,3-aminophenylbutane, and the like, showed favorable reactivities.

TABLE 8 Relative Compound Activity (%) (R)-1-Phenylethylamine 100(R)-3,4-Dimethoxyaminopropane 30 1-Methylheptylamine 47 2-Heptylamine 371-Methylpropylamine 1.2 Benzylamine 0.8 (S)-2-Phenylglycinol 1803-Aminophenylbutane 84 L-Phenylalaninol 0.8

EXAMPLE 12

Using the purified enzyme obtained in Example 9, reactivities to variouscarbonyl compounds using (R)-1-phenylethylamine as an amino donor wereevaluated. 50 μl of a solution of the purified enzyme was added to 200μl of a substrate solution (0.1 M Tris-HCl buffer (pH 8.5), 25 mM(R)-1-phenylethylamine, 0.1 mM pyridoxal phosphate, and 25 mM variouscarbonyl compounds), and the components were reacted at 30° C. for 1 to3 hours. The reaction tube was transferred into boiling water to stopthe reaction, and, thereafter, the reaction mixture was dilutedfive-folds with methanol. This diluted solution was analyzed byhigh-performance liquid chromatography using fine pack C18-5 column andusing methanol/water (20:80) as an eluent solution to quantitativelyanalyze acetophenone formed by the reaction. The results thereof areshown in Table 9 in terms of relative activity in the case of usingpyruvic acid as a substrate. It is apparent from the Table 9 thatoxalacetic acid, phenoxy-2-propanone, and the like also showed favorablereactivities.

TABLE 9 Relative Compound Activity (%) Pyruvic acid 100 Oxalacetic acid87 Glyoxylic acid 4 2-Ketobutyric acid 7 Ethyl pyruvate 27 Ethylacetoacetate 4 2-Decane 3 4-Methoxyphenyl acetone 141-(3,4-Dimethoxyphenyl)-2-propanone 7 Benzyl acetone 34-(4-Methoxyphenyl)-2-butanone 7 1-Phenyl-2-butanone 1 Phenylacetaldehyde 13 Ethyl benzoyl acetate 2 Phenoxy-2-propanone 83 Diacetyl10 1-Methoxy-2-propanone 16 1-Tetralone 1 2-Acetylpyridine 193-Acetylpyridine 8 4-Acetylpyridine 27 3-Acetoxypyridine 12-Acetylpyrazine 17 2-Acetylfuran 2 2-Acetylthiophene 1 2-Acetylthiazole10

EXAMPLE 13

The Arthrobacter species KNK168 strain was cultured in the same manneras in Example 7, and the cells were separated from 1 ml of the culture.The cells were suspended in 1 ml of a reaction mixture containing a 0.1M Tris-hydrochloric acid buffer (pH 8.5), 100 mM (R)-1-phenylethylamine,100 mM methoxy-2-propanone, and 0.1% of sodium dodecyl sulfate. Thecomponents were reacted at 30° C. for 20 hours with stirring. As aresult of the analysis of the reaction mixture, methoxy-2-propanone, thesubstrate, almost disappeared and converted to 2-amino-1-methoxypropane,and its optical purity was 99.4% ee ((R)-form).

EXAMPLE 14

The Arthrobacter species KNK168 strain was cultured in the same manneras in Example 13, and the cells were separated from 1 ml of the culture.The cells were suspended in 1 ml of a reaction mixture containing a 0.1M Tris-hydrochloric acid buffer (pH 8.5), 50 mg of racemic2-amino-1-methoxypropane, 37.5 mg of pyruvic acid, and 0.1% of sodiumdodecyl sulfate. The components were reacted at 30° C. for 24 hours withstirring. As a result of the analysis after the reaction, 23.4 mg of2-amino-1-methoxypropane remained, and its optical purity was 95% ee((S)-form).

EXAMPLE 15

By carrying out the same reaction as in Example 14 using 200 mM racemic2-amino-1-methoxypropane, and 150 mM n-butylaldehyde or propionaldehyde,the components were reacted at 30° C. for 20 hours. As a result of theanalysis thereof, the (S)-form of 2-amino-1-methoxypropane wasconcentrated, and the ratio of the (S)-form was 76% in a case of usingn-butylaldehyde, and 58% in a case of using propionaldehyde.

EXAMPLE 16

The influence of pH on the activity was evaluated by using the purifiedenzyme obtained in Example 9. 0.1 ml of an enzyme solution which wasdiluted suitably beforehand was added to 0.9 ml of a solution containing20 μmol of 1-(3,4-dimethoxyphenyl)-2-propanone, 20 μmol of(R)-1-phenylethylamine, and 1 μmol of pyridoxal phosphate, of which thepH was adjusted using the buffer described as follows. The componentswere reacted at 30° C. for 1 hour. The used buffers were an acetic acidbuffer (CH₃COONa being used as the abbreviation; pH 4 to 6), a potassiumphosphate buffer (KPB; pH 5 to 8), and a Tris-hydrochloric acid buffer(Tris-HCl; pH 7 to 10), all of these buffers having final concentrationsof 0.1 M. The formed (R)-DMA in the reaction mixture after the reactionwas quantitatively analyzed by high-performance liquid chromatography.The relative activity at each pH, relative to the activity at pH 8.5 asbeing 100%, is shown in FIG. 1. It was found that the optimum pH of thisenzyme is in the neighborhood of pH 8.5 (pH 7.5 to 9.0).

EXAMPLE 17

The influence of the pH on the stability of the enzyme was evaluated byusing the purified enzyme obtained in Example 9. After the pH of theenzyme solution was adjusted by HCl or NaOH, the reaction mixture wasincubated at 20° C. for 23 hours. Thereafter, in the same manner as inExample 16, the components were reacted at pH 8.5, and the formed(R)-DMA was quantitatively analyzed. The results in which the relativeactivities of the treated samples at each pH, relative to the activityof the untreated enzyme solution as being 100%, are shown in FIG. 2. Itwas found that this enzyme was most stable at a pH in the neighborhoodof 7.

EXAMPLE 18

The Arthrobacter species KNK168 strain was subjected to shaking culturein the same manner as in Example 6 using (RS)-1-methylpropylamine as anenzyme inducer. The cells harvested from one liter of the culture weresuspended in one liter of a 0.1 M Tris-hydrochloric acid buffer (pH8.5), and 30 g of 1-(3-trifluoromethylphenyl)-2-propanone, 18 g of(R)-1-phenylethylamine, and 44 g of oleic acid were added thereto. Thecomponents were reacted at 30° C. for 40 hours with stirring. After thereaction, the resulting mixture was subjected to extraction anddistillation in the same manner as in Example 6, to give 19.5 g of(R)-1-(3-trifluoromethylphenyl)-2-aminopropane. The optical purity was100% ee, and the yield was 65%.

INDUSTRIAL APPLICABILITY

According to the present invention, it is made possible to easilyprepare at a high yield the optically active (R)-amino compounds and thelike having an aryl group and the like at their 1-position, which havebeen conventionally difficult to prepare.

IDENTIFICATION OF DEPOSITED MICROORGANISM

(1) Name and Address of Depository Organization The Ministry ofInternational Trade and Industry, National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology 1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan (zip code 305)

(2) Date of Deposit Sep. 8, 1995 (date of original deposit)

(3) Accession Number FERM BP-5228

1 1 12 PRT Arthrobacter sp. 1 Glu Ile Val Tyr Thr His Asp Thr Gly LeuAsp Tyr 1 5 10

What is claimed is:
 1. A method for preparing an optically active(R)-amino compound comprising the step of stereoselectively carrying outamino group transfer by action of an (R)-form-specific transaminase inthe co-presence of a ketone compound (amino acceptor) and an aminocompound (amino donor), wherein said amino acceptor is a compoundrepresented by the following general formula (I):

wherein n is 0 to 5; m is 0 or 1; and X represents an unsubstituted arylgroup having 6 to 14 carbon atoms; an aryl group having 6 to 14 carbonatoms and having one or more substitutents selected from the groupconsisting of an alkyl group having 4 to 15 carbon atoms, a hydroxylgroup, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom,a nitro group, a carboxyl group, and a trifluoromethyl group; asubstituted phenyl group selected from the group consisting of2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2,4-dimethoxyphenyl,3,4-dimethoxyphenyl and 3-trifluoromethylphenyl; a methoxyl group; apyridyl group; a pyrazinyl group; a thienyl group; a furyl group; or athiazolyl group; and wherein said amino donor is a compound of anachiral form, a racemic form, or an (R)-form represented by the generalformula (II):

wherein X₁ and X₂ independently represent a hydrogen atom; astraight-chain alkyl group having 1 to 10 carbon atoms; a branched alkylgroup having 5 to 12 carbon atoms; an unsubstituted aryl group having 6to 14 carbon atoms; an aryl group having 6 to 14 carbon atoms and havingone or more substitutents selected from the group consisting of an alkylgroup having 1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, achlorine atom, a bromine atom, an iodine atom, a nitro group, and acarboxyl group; an aralkyl group having 7 to 16 carbon atoms and havingone or more substitutents selected from the group consisting of an alkylgroup having 1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, achlorine atom, a bromine atom, an iodine atom, a nitro group, and acarboxyl group; or a hydroxymethyl group, thereby giving an opticallyactive (R)-amino compound represented by the general formula (IV):

wherein n, m, and X have the same definitions as those of n, m, and X inthe general formula (I), respectively.
 2. The method for preparing anoptically active (R)-amino compound according to claim 1, wherein in thegeneral formula (II), X₁ is an alkyl group having 2 to 10 carbon atoms,a phenyl group, or a naphthyl group, and X₂ is an alkyl group having 1or 2 carbon atoms.
 3. The method for preparing an optically active(R)-amino compound according to claim 1, wherein the amino donor is(R)-1-phenylethylamine, (R)-1-naphthylethylamine,(R)-1-methylpropylamine, (R)-2-aminopentane, (R)-2-amino-1-propanol,(R)-1-methylbutylamine, (R)-1-phenylmethylamine,(R)-1-amino-1-phenylethanol, (R) -2-amino-2-phenylethanol, (R)-3-aminoheptane, (R)-1-amino-3-phenylpropane,(R)-2-amino-4-phenylbutane, (R)-2-amino-3-phenylpropanol,(R)-3,4-dimethoxyaminopropane, (R)-1-methylheptylamine, benzylamine,(S)-2-phenylglycinol-3-aminophenylbutane, L-phenylalaninol,(R)-2-amino-1-methoxypropane, or a racemic compound thereof.
 4. Themethod for preparing an optically active (R)-amino compound according toclaim 1, wherein in the general formula (I) and the general formula(IV), n and m are n=1 and m=0.
 5. The method for preparing an opticallyactive (R)-amino compound according to claim 1, wherein the aminoacceptor and the amino donor are brought into contact with a culture ofmicroorganisms, separated bacterial cells, treated bacterial cells, orimmobilized bacterial cells which produce (R)-form specifictransaminases.
 6. The method for preparing an optically active (R)-aminocompound according to claim 1, wherein the amino acceptor and the aminodonor are brought into contact with cell-free extracts ofmicroorganisms, crudely purified enzymes, purified enzymes, orimmobilized enzymes which produce (R)-form specific transaminases. 7.The method for preparing an optically active (R)-amino compoundaccording to claim 1, wherein the microorganism for producing thetransaminase is a microorganism belonging to the genus Arthrobacter. 8.The method for preparing an optically active (R)-amino compoundaccording to claim 7, wherein the microorganism belonging to the genusArthrobacter is Arthrobacter species (Arthrobacter sp.) KNK168 (FERMBP-5228).
 9. The method for preparing an optically active (R)-aminocompound according to claim 1, wherein when culturing the microorganismfor producing the transaminase, one or more members selected from thegroup consisting of (RS)-1-methylpropylamine, (RS)-1-phenylethylamine,(RS)-1-methylbutylamine, (RS)-3-amino-2,2-dimethylbutane,(RS)-2-amino-1-butanol, and (R)- or(RS)-1-(3,4-dimethoxyphenyl)aminopropane are added to a medium as aninducer for the enzyme.
 10. The method for preparing an optically active(R)-amino compound according to claim 1, wherein a reaction at a pH ofnot less than 5 and not more than 12 is carried out in the amino grouptransfer reaction.
 11. The method for preparing an optically active(R)-amino compound according to claim 1, wherein a surfactant or a fattyacid is added as a reaction accelerator upon reaction in the amino grouptransfer reaction.
 12. A method for preparing an optically active(S)-amino compound, comprising the step of stereoselectively carryingout amino group transfer reaction by action of an (R)-form-specifictransaminase to an amino compound of a racemic form represented by thegeneral formula (V) in the presence of a ketone compound (aminoacceptor) represented by the general formula (III), to give an opticallyactive (S)-amino compound represented by the general formula (VI):

wherein X₁ and ₂ independently represent a hydrogen atom; astraight-chain alkyl group having 1 to 10 carbon atoms; a branched alkylgroup having 5 to 12 carbon atoms; an unsubstituted aryl group having 6to 14 carbon atoms; an aryl group having 6 to 14 carbon atoms and havingone or more substituents selected from the group consisting of an alkylgroup having 1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, achlorine atom, a bromine atom, an iodine atom, a nitro group, and acarboxyl group; an aralkyl group having 7 to 16 carbon atoms and havingone or more substituents selected from the group consisting of an alkylgroup having 1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, achlorine atom, a bromine atom, an iodine atom, a nitro group, and acarboxyl group; or a hydroxymethyl group or a hydroxyethyl group; andwherein n is 0 to 5; m is 0 or 1; and X represents an unsubstituted arylgroup having 6 to 14 carbon atoms; an aryl group having 6 to 14 carbonatoms and having one or more substituents selected from the groupconsisting of an alkyl group having 4 to 15 carbon atoms, a hydroxylgroup, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom,a nitro group, a carboxyl group, and a trifluoromethyl group; or amethoxyl group.